Fig. 4

ERG is differentially phosphorylated by BMP10 in HUVECs. A HUVECs were stimulated with 10 ng/mL BMP10 or not (NS) for 30 min. Cell extracts were subjected to immunoprecipitation (IP) using ERG antibody, followed by WB using pan phosphoserine (pSer) and ERG antibodies. Total serine phosphorylation of ERG was quantified and normalized to total ERG levels. IgG represents lysates subjected to IP using isotype control antibody. Whole cell extracts (input) were subjected to WB analysis using antibodies against pSMAD1/5, ERG and HSP90. Data are presented as mean folds (BMP10-vs-NS) ± SEM of n = 4 independent experiments.*P < 0.05 using Kolmogorov–Smirnov test. B HUVECs were stimulated with 10 ng/mL BMP10, or VEGF (40 ng/mL), BMP10 + VEGF (10 and 40 ng/mL, respectively) or not (NS) for 30 min. Cell extracts were subjected to IP using ERG antibody, followed by WB using antibodies against ERG-Ser215 and ERG. Quantification of ERG phosphorylation reflects the normalized signal for the pERG-Ser215 to total ERG from each sample. IgG represents lysates subjected to IP using isotype control antibody. Whole cell extracts (input) were subjected to WB analysis using antibodies against pERK1/2-Thr202/Tyr204, ERK1/2, ERG, pSMAD1/5, and HSP90 (loading control). Data are presented as mean folds (BMP10, VEGF or VEGF + BMP10-vs-NS) ± SEM of at least 5 independent experiments. Statistical analyses were performed using two-way ANOVA followed by Sidak's multiple comparisons post-test. *P < 0.05